The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modern biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. See, e.g., “DNA Sequencing,” Current Protocols In Molecular Biology, Vol. 1, Chapter 7 (1995). The sequencing of several important genomes has already been completed (e.g., yeast, E. coli), and work is proceeding on the sequencing of other genomes of medical and agricultural importance (e.g., human, C. elegans, Arabidopsis). In the medical context, it will be necessary to “re-sequence” the genome of large numbers of human individuals to determine which genotypes are associated with which diseases. Such sequencing techniques can be used to determine which genes are active and which are inactive, either in specific tissues, such as cancers, or more generally in individuals exhibiting genetically influenced diseases. The results of such investigations can allow identification of the proteins that are good targets for new drugs or identification of appropriate genetic alterations that may be effective in genetic therapy. Other applications lie in fields such as soil ecology or pathology where it would be desirable to be able to isolate DNA from any soil or tissue sample and use probes from ribosomal DNA sequences from all known microbes to identify the microbes present in the sample.
The conventional sequencing of DNA using electrophoresis is typically laborious and time consuming. Various alternatives to conventional DNA sequencing have been proposed. One such alternative approach, utilizing an array of oligonucleotide probes synthesized by photolithographic techniques is described in Pease et al., “Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,” Proc. Natl. Acad. Sci. USA 91:5022-5026 (1994). In this approach, the surface of a solid support modified with photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3′ activated deoxynucleoside, protected at the 5′ hydroxyl with a photolabile group, is then provided to the surface such that coupling occurs at sites that had been exposed to light. Following capping and oxidation, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5′ protected activated deoxynucleoside base is presented to the surface. The selective photodeprotection and coupling cycles are repeated to build up levels of bases until the desired set of probes is obtained. It may be possible to generate high density, miniaturized arrays of oligonucleotide probes using such photolithographic techniques, wherein the sequence of the oligonucleotide probe at each site in the array is known.
These probes can then be used to search for complementary sequences on a target strand of DNA, with detection of the target that has hybridized to particular probes accomplished by the use of fluorescent markers coupled to the targets and inspection by an appropriate fluorescence scanning microscope. A variation of this process using polymeric semiconductor photoresists, which are selectively patterned by photolithographic techniques, rather than using photolabile 5′ protecting groups, is described in McGall et al., “Light-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists,” Proc. Natl. Acad. Sci. USA 93:13555-13560 (1996), and McGall et al., “The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates,” J. Am. Chem. Soc. 119:22:5081-5090 (1997).
A disadvantage of both of these approaches is that four different lithographic masks are needed for each monomeric base, and the total number of different masks required are thus four times the length of the DNA probe sequences to be synthesized. The high cost of producing the many precision photolithographic masks that are required, and the multiple processing steps required for repositioning of the masks for every exposure, contribute to relatively high costs and lengthy processing times.
A similar problem exists for synthesis of diverse sequences of other types of oligomers such as polypeptides, which is useful for determining binding affinity in screening studies. For example, Piffling et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) discloses methods of forming vast arrays of peptides using light-directed synthesis techniques. However, the large number of lithographic masks needed in the synthesis makes the fixed cost for this process relatively high and the processing time lengthy.
A patterning process described in Cerrina et al., PCT Application No. WO 99/42813 overcomes the above problems. With this patterning process, an image is projected onto an activate surface of a substrate for oligomer synthesis utilizing an image former that includes a light source that provides light to a micromirror device including an array of electronically addressable micromirrors. The substrate is activated in a defined pattern and monomers are coupled to the activated sites, with further repeats until the elements of a two-dimensional array on the substrate have an appropriate monomer bound thereto. The micromirror arrays can be controlled in conjunction with an oligomer synthesizer to control the sequencing of images presented by the micromirror array in coordination with the reagents provided to the substrate. The patterning process eliminated the requirement of lithographic masks for selectively illuminating certain oligomer synthesis positions.
In an instrument for the synthesis of nucleic acid probes using light, strict control of the light in the instrument has proven to be a critical parameter. Light that is misdirected, inadvertently reflected or otherwise directly randomly inside the instrument, herein referred to as “stray light,” can adversely affect the overall accuracy and fidelity of the arrays made by the instrument. Excess stray light can lead to the de-protection of areas of the array other than the ones intended to be de-protected, and thus cause errors in the synthesis of probes. This problem cannot be well-controlled in a photolithographic process, where the use of masks interposed between the light source and the array synthesis cell inherently causes refracted light in some amount to be direct where it is not intended. However, the development of the maskless array synthesizer permits the level of stray light in the instrument to be minimized to an extent that was not possible before.